1Núñez- Torrón C, et al. J Clin Pathol 2021;0:1–7. doi:10.1136/jclinpath-2020-207337

Secondary haemophagocytic lymphohistiocytosis in COVID-19: correlation of the autopsy findings of bone marrow haemophagocytosis with HScoreClaudia Núñez- Torrón ,1 Ana Ferrer- Gómez,2 Esther Moreno Moreno,2 Belen Pérez- Mies,2,3,4 Jesús Villarrubia,1 Sandra Chamorro,5 Javier López- Jiménez,1,3 J Palacios,2,3,4 Miguel Piris- Villaespesa,1 Mónica García- Cosío2,3,4

Original research

To cite: Núñez- Torrón C, Ferrer- Gómez A, Moreno Moreno E, et al. J Clin Pathol Epub ahead of print: [please include Day Month Year]. doi:10.1136/jclinpath-2020-207337

1Hematology, Hospital Ramón y Cajal, Madrid, Spain2Pathology, Hospital Ramón y Cajal, Madrid, Spain3Faculty of Medicine, University of Alcalá de Henares, Madrid, Spain4CIBER- ONC, Instituto de Salud Carlos III, Madrid, Spain5Infectious Diseases, Hospital Ramón y Cajal, Madrid, Spain

Correspondence toDr Mónica García- Cosío, Pathology, Hospital Ramón y Cajal, Madrid 28034, Spain; monica. garciacosio@ salud. madrid. org

CN- T and AF- G are joint first authors.MP- V and MG- C are joint senior authors.

Received 11 December 2020Revised 19 January 2021Accepted 15 February 2021

© Author(s) (or their employer(s)) 2021. No commercial re- use. See rights and permissions. Published by BMJ.

ABSTRACTBackground Secondary haemophagocytic lymphohistiocytosis (sHLH) is characterised by a hyper activation of immune system that leads to multiorgan failure. It is suggested that excessive immune response in patients with COVID-19 could mimic this syndrome. Some COVID-19 autopsy studies have revealed the presence of haemophagocytosis images in bone marrow, raising the possibility, along with HScore parameters, of sHLH.Aim Our objective is to ascertain the existence of sHLH in some patients with severe COVID-19.Methods We report the autopsy histological findings of 16 patients with COVID-19, focusing on the presence of haemophagocytosis in bone marrow, obtained from rib squeeze and integrating these findings with HScore parameters. CD68 immunohistochemical stains were used to highlight histiocytes and haemophagocytic cells. Clinical evolution and laboratory parameters of patients were collected from electronic clinical records.Results Eleven patients (68.7%) displayed moderate histiocytic hyperplasia with haemophagocytosis (HHH) in bone marrow, three patients (18.7%) displayed severe HHH and the remainder were mild. All HScore parameters were collected in 10 patients (62.5%). Among the patients in which all parameters were evaluable, eight patients (80%) had an HScore >169. sHLH was not clinically suspected in any case.Conclusions Our results support the recommendation of some authors to use the HScore in patients with severe COVID-19 in order to identify those who could benefit from immunosuppressive therapies. The presence of haemophagocytosis in bone marrow tissue, despite not being a specific finding, has proved to be a very useful tool in our study to identify these patients.

INTRODUCTIONThe outbreak of the novel coronavirus pandemic is supposing a medical challenge worldwide. The main clinical features of COVID-19 vary from asymp-tomatic or mild symptoms such as fever, cough and myalgia to severe pneumonia with acute respira-tory distress syndrome (ARDS), requiring at times ventilatory support.1–5 Severe patients also display signs of high systemic inflammatory response with altered pattern of inflammatory chemokines and cytokines and high ferritin levels, known as cytokine storm.4 6IL-1/IL-6 pathway dysregulation seems to

play a crucial role in the pathogenesis of the severe complications of patients with COVID-19, as has been suggested by the favourable evolution of some patients treated with their receptor antagonists.7 8

Secondary haemophagocytic lymphohistio-cytosis (sHLH), cytokine release syndrome and macrophage activation syndrome are overlap-ping syndromes characterised by an activation of lymphocytes and macrophages with a subsequent excessive immune response, cytokine storm and haemophagocytosis, which leads to multiorgan damage.9–12The most frequent triggers of this syndrome are infections and malignancies. Among viral infections, the most frequent causative agent is Epstein- Barr virus, but other viruses like respiratory syncytial virus, rotavirus and adenovirus infections have been reported.13–15

Clinical presentation of sHLH consists of fever, hepatosplenomegaly, cytopenias, acute liver failure, high ferritin and CD25 soluble (sCD25), coagulop-athy, dermatologic manifestations as Kawasaki- like syndrome and neurological symptoms.10 Around 50% of patients develop respiratory symptoms.15 Diagnosis of sHLH is based on clinical and labo-ratory criteria. Until recently, the criteria were extrapolated from the primary haemophago-cytic lymphohistiocytosis (HLH2004 criteria).16 However, in 2014, Fardet et al proposed a new score validated in patients with reactive HLH, called the HScore.17 It is based on nine variables: three clinical features, five laboratory parameters and one pathological finding, which is the presence of haemophagocytosis in bone marrow.

In accordance, some authors suggest that the immune response seen in COVID-19 could mimic sHLH and, therefore, recommend using the HScore to identify patients who could benefit from immu-nosuppressive therapies.18

The presence of haemophagocytosis in bone marrow could raise the possibility of sHLH in patients with severe COVID-19. Wood et al, based on intensive care unit (ICU) patients’ study, have considered that HScore has limited application in severe patients with COVID-19. Nevertheless, they calculated HScore without bone marrow biopsy.19

Various autopsy series on patients with COVID-19 have begun to emerge in the literature. Only few of them include the study of bone marrow tissue for the presence of haemophagocytosis. Some of these studies, based on the hypothesis above described,

on Novem

ber 2, 2021 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jclinpath-2020-207337 on 15 M

arch 2021. Dow

nloaded from

2 Núñez- Torrón C, et al. J Clin Pathol 2021;0:1–7. doi:10.1136/jclinpath-2020-207337

Original research

correlate this finding, along with HScore, with a possible hyper-inflammatory status and hypercytokinemia/sHLH, reporting contradictory results about the presence of haemophagocytosis and the convenience of using the HScore in these patients due to a potential lack of sensitivity.20–25 In order to ascertain the existence of sHLH in patients with COVID-19, we report the histological findings from 16 COVID-19 autopsies, one of the largest series published until now that focuses on the presence of haemophagocytosis in bone marrow integrating these findings with clinical and laboratory criteria for sHLH diagnosis.

METHODSPatient selectionWe performed a single- centre retrospective analysis in 16 patients with COVID-19 disease in a tertiary care hospital. We considered as confirmed cases the patients with compatible clin-ical symptoms or a suggestive chest image and a positive reverse transcription- PCR for SARS- Cov-2 nucleic acid on upper respi-ratory swap.26

Clinical and laboratory dataWe calculated the HScore for all patients. The HScore is freely available online (http:// saintantoine. aphp. fr/ score/). As previous recommendations for this score, we used a cut- off point of 169 points, corresponding to a sensitivity of 93% and a specificity of 86% for the diagnosis of sHLH.17 Clinical evolution and labora-tory parameters were collected from electronic clinical records. For the laboratory parameters, the value corresponding to the maximum score during evolution was considered. Organomegaly was obtained from physical exploration or from radiological findings. Missing data were scored as 0 points. The presence of haemophagocytosis was assessed postmortem in bone marrow tissue from autopsies, as explained below.

We did not calculate the HLH 2004 score because of the sCD25 and the natural killer (NK)- cell activity were not avail-able in any patients.

Autopsy procedureAll autopsies were performed between 19 April and 4 June, when clinicians requested them with the consent of relatives, in patients with severe COVID-19 with unexpected unfavourable clinical course. They were conducted in a negative pressure room using personal protective equipment and performed according to a security protocol as previously reported.27 We collected tissue from each organ according to our autopsy protocol,27 which includes haematopoietic tissue. Collected tissues were fixed for 24 hours in 10% buffered formalin. After fixation, rib tissue was decalcified with formic acid for 24 hours before embedding in paraffin blocks. Multiple unstained 3–5 µm- thick sections were cut for H&E stain and immunohistochemistry. H&E staining was applied according to the standard protocols.

ImmunohistochemistryTo identify the presence and the architectural distribution of the different haematopoietic series and lymphoid populations, immunohistochemistry was performed using a routine panel of antibodies against CD20 (clone L26, Agilent), CD3 (polyclonal, Agilent), CD4 (clone 4B12, Agilent), CD8 (clone C8/144B, Agilent), CD68 (clone PG- M1, Agilent), glycophorin C (clone ret40f, Agilent), CD61 (clone Y2/51, Agilent) and myeloperox-idase (polyclonal, Agilent). All of them were performed on an OMNIS (Agilent) automated stainer, except for CD71 (MRQ-48, Roche), which was performed on the Benchmark Ultra Ventana

systems (Roche). CD68 immunohistochemical stains were used to highlight the histiocytes and haemophagocytic cells. Double staining glycophorin C/CD68 was performed in order to identify more precisely haemophagocytic cells.

Bone marrow histiocytic quantificationOnly histiocytes showing engulfment of one or more nucleated host blood cells, including plasma cells and lymphocytes, were counted as haemophagocytosis. Moreover, morphological evalu-ation of histiocytic hyperplasia with haemophagocytosis (HHH) was graded in bone marrow tissue based on Suster et al score28 such as: mild HHH (haemophagocytosis was present only when searched for in several high- power fields (HPF)), moderate (haemophagocytosis was present in one to three cells per HPF) and severe (haemophagocytosis was present more than three cells per HPF). This classification considers as pathological HHH only those cases demonstrating moderate to severe haemophago-cytic activity. Nevertheless, since the HScore requires only the presence of haemophagocytosis in bone marrow to meet this parameter,17 we used this classification in order to establish a possible association between sHLH diagnosis and HHH degree. We also calculated the percentage of histiocytes in each sample by counting the number of histiocytes stained with CD68 in 500 bone marrow cells. In normal conditions, the histiocytic cellu-larity in bone marrow is inconspicuous. Histiocytic hyperplasia was considered in our study when the percentage was 5% or superior.

Moreover, following the Gars et al study,29 we searched for the presence of multiple nucleated cells within a single haemophago-cyte in each case.

RESULTSClinical findingsSixteen patients were collected. All of them had positive SARS- CoV-2 PCR. The median age of our cohort was 64·5 years (range 57–73). The 75% of patients were men. The symptoms at diagnosis were fever (87·5%), cough (56·3%), dyspnoea (37·5%), gastrointestinal symptoms (31·3%) and asthenia (18.8%). Other less common symptoms were chest pain (12·5%), anosmia (6) and odynophagia (6.3%). All patients suffered from ARDS and 14 (87·5%) required orotracheal intubation and admission to an ICU. Nine patients (56·3%) suffered acute renal injury and 18.8% underwent dialysis treatment.

The median time from COVID-19 diagnosis to death was 33 days (range 24–42). From the 16 patients, 11 (68·8%) nega-tivised SARS- Cov-2 PCR in a median 23 days (range 20–36). In three patients, control sequential SARS- Cov-2 PCR was not performed.

Following the clinical protocol, lopinavir/ritonavir was prescribed in 11 patients (68·8%), hydroxychloroquine in 14 patients (87·5%) and azithromycin in 12 patients (75%). Corti-costeroids were administered in 14 patients (87·5%) and tocili-zumab in 13 patients (81·3%). We reviewed blood, respiratory and urinary cultures. In nine patients (56·3%), other infectious pathogens were isolated. These patients received concomitant antimicrobial treatment.

We reviewed the prior relevant comorbidities to COVID-19 diagnosis, which were arterial hypertension in six patients (37·5%), hypercholesterolemia in five patients (31·2%), cardio-vascular disease in five patients (31·2%), sleep apnoea syndrome in two patients (12·5%), cognitive impairment in two patients (12·5%) and hepatitis C virus hepatopathy in one patient (6·3%). Three patients had oncohematological history and only

on Novem

ber 2, 2021 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jclinpath-2020-207337 on 15 M

arch 2021. Dow

nloaded from

3Núñez- Torrón C, et al. J Clin Pathol 2021;0:1–7. doi:10.1136/jclinpath-2020-207337

Original research

one of them was in treatment with intensive chemotherapy due to myeloproliferative neoplasm in blast phase.

Autopsy findingsPathological autopsy findings are described in table 1.

Regarding to bone marrow tissue, HHH was observed in all cases in varying degrees (Figure 1).

Histiocytic hyperplasia was easily observed in all cases but one, with percentages, ranged from 7% to 13%.

Eleven patients (68.7%) displayed moderate HHH, 3 patients (18·7%) displayed severe HHH and 2 (12·5%) displayed mild HHH. The maximum number of haemophagocytosis was objec-tified in case 12 (4·7 haemophagocytosis/HPF). These specific values are reflected in table 2, along with the remaining param-eters of HScore.

Immunohistochemical study also demonstrated CD8+ predominance lymphocytosis as a common finding in all cases. The histological and immunohistochemical features in bone marrow tissue were very similar in all the cases. The overall cellularity was increased with left shift deviation from granulo-cyte series and hyperplasia of megakaryocytic series with reac-tive changes was also observed.

The presence of more than one nucleated cell within a single haemophagocyte was observed in all but two cases (cases 13 and 16). These two cases were the ones that displayed mild HHH.

Lung tissue from each lobe was collected. In all cases, lungs had a consolidated appearance, especially the lower lobes. Histologically, the main finding in lung parenchyma was diffuse alveolar damage (DAD), both in its proliferative phase (10/16 patients) and in its exudative phase (6/16 patients). Four patients also showed DAD in advanced fibrous phase. One patient showed interstitial lymphoid pneumonitis. In all cases, primary cause of death was attributed to the severe lung damage.

Spleen tissue could be evaluated only in 13 patients, due to autolytic changes in three of them. The main finding in spleen tissue was depletion of white pulp, seen in 11/13 patients. Histio-cytic hyperplasia was observed in 10 patients. Haemophago-cytosis of nucleated cells was seen in four patients. CD8+ predominance lymphocytosis was also observed in splenic tissue, most of them in a lower degree compared with bone marrow tissue. Only one case (case 9) showed severe CD8+ lymphocy-tosis. Regarding other findings, extramedullary haematopoiesis was evidenced in five patients.

Table 1 Pathological findings in lung, spleen and lymph node

Case Lung tissue findings Spleen tissue findings Lymph node tissue findings

1 DAD (fibrous phase) Necrotic tissue Not collected

2 DAD (exudative phase) Necrotic tissue Not collected

3 DAD (exudative phase) Focal necrosis. Histiocytic hyperplasia Not collected

4 DAD (proliferative phase) Depletion of the white pulp. Haematopoiesis. Histiocytic hyperplasia. Haemophagocytosis.

Not collected

5 DAD (proliferative phase) Histiocytic hyperplasia. Erytrophagocytosis. Plasmocytosis. Erythrophagocytosis.

6 DAD (proliferative phase) Widely necrotic. Depletion of the white pulp. Lymphoid depletion. Erythrophagocytosis.

7 DAD (proliferative phase) Depletion of the white pulp. Histiocytic hyperplasia. Severe haemophagocytosis.

Not collected

8 DAD (exudative phase)+acute bronchopneumonia

Depletion of the white pulp. Haematopoiesis. Histiocytic hyperplasia. Lymphoid depletion. Plasmocytosis. Erythrophagocytosis.

9 DAD (proliferative phase) Haemorrhage. Depletion of the white pulp. Histiocytic hyperplasia. Erythrophagocytosis.

Lymphoid depletion. Plasmocytosis. Erythrophagocytosis.

10 DAD (proliferative phase) Focal necrosis. Haemorrhage. Depletion of the white pulp. Not collected

11 DAD (proliferative phase) Focal necrosis. Haemorrhage. Depletion of the white pulp. Haematopoiesis. Not collected

12 Interstitial lymphoid pneumonitis Depletion of the white pulp. Histiocytic hyperplasia. Haemophagocytosis. Not collected

13 DAD (exudative phase) Depletion of the white pulp. Histiocytic hyperplasia. Haemophagocytosis. Lymphoid depletion. Erythrophagocytosis.

14 DAD (exudative, proliferative and fibrous phase).

Depletion of the white pulp. Haematopoiesis. Histiocytic hyperplasia. Not collected

15 DAD (exudative, proliferative and fibrous phase)

Depletion of the white pulp. Haematopoiesis. Histiocytic hyperplasia. Lymphoid depletion

16 DAD (proliferative and fibrous phase) Necrotic tissue Not collected

DAD, diffuse alveolar damage.

Figure 1 Bone marrow pathological findings. (A) (H&E stain, 40×) High- power view of haemapoietic cells in bone marrow. (B) (CD68 stain. 10×) Low- power view of bone marrow with histiocytic hyperplasia with haemophogocytosis. (C) (CD68 stain, 40×) High power view of severe HHH in born marrow. (D) (CD68 stain, 100x) high- power view of haemophagocytosis images. HHH, hyperplasia with haemophagocytosis.

on Novem

ber 2, 2021 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jclinpath-2020-207337 on 15 M

arch 2021. Dow

nloaded from

4 Núñez- Torrón C, et al. J Clin Pathol 2021;0:1–7. doi:10.1136/jclinpath-2020-207337

Original research

Tabl

e 2

HSco

re p

aram

eter

s an

d bo

ne m

arro

w p

atho

logi

cal fi

ndin

gs

Pati

ent

Basa

l IS

*H

epat

omeg

aly

Sple

nom

egal

yM

axim

um b

ody

T (°

C)Lo

wes

t H

b*Lo

wes

t W

BC†

Low

est

plat

elet

s†M

axim

um

ferr

itin

‡M

axim

um

trig

lyce

rid§

Low

est

fibri

noge

n¶M

ax

GO

T**

Hae

mop

hago

cyto

sis

in B

MN

° hi

stio

cyte

s††

Hae

mop

hago

cyt

imag

es p

er H

PFH

Scor

e po

ints

‡‡

%

Prob

abili

tyH

Scor

e

1N

oN

oN

o<

38.4

°C8.

86.

448

.427

004.

817

021

5Ye

s60

(12%

)1.

320

7/33

792

2N

oN

oN

o<

38.4

°C10

.48.

4817

7N

AN

A53

048

Yes

50 (1

0%)

2.3

54/2

230.

09

3N

oN

oN

o>

38.4

°C<

39.4

°C7.

32.

9671

.919

886.

4518

010

1Ye

s36

(7%

)1.

919

6/33

785

4N

oN

oN

o>

38.4

°C<

39.4

°C7.

92.

417

.6N

AN

A14

011

6Ye

s50

(10%

)1.

415

1/22

326

.3

5N

oN

oN

o<

38.4

°C7

2.35

4114

364.

1616

074

Yes

67 (1

3%)

3.2

182/

337

70.9

7

6N

oN

oN

o>

38.4

°C<

39.4

°C8.

14.

6953

2163

NA

100

82Ye

s55

(11%

)3.

218

6/27

375

.8

7N

oN

oN

o>

38.4

°C<

39.4

°C8.

72.

810

679

5N

A12

054

Yes

55 (1

1%)

1.4

151/

273

26.3

8Ye

sN

oN

o>

38.4

°C<

39.4

°C10

.12.

817

0N

AN

A36

022

Yes

50 (1

0%)

1.3

86/2

230.

63

9N

oN

oN

o>

38.4

°C<

39.4

°C7.

64.

0760

.916

895.

116

067

Yes

62 (1

2%)

1.7

215/

337

94.9

7

10N

oN

oN

o≥

39.4

°C8.

34.

0279

.751

69.

3114

012

4Ye

s50

(10%

)2.

221

2/33

794

11N

oN

oN

o>

38.4

°C<

39.4

°C7.

68.

712

014

541.

236

058

Yes

59 (1

2%)

1.8

87/3

370.

67

12N

oN

oN

o≥

39.4

°C12

.510

.431

.671

40.

4916

087

Yes

60 (1

2%)

4.7

133/

337

10.4

13Ye

sYe

sYe

s≥

39.4

°C6.

50.

018

2494

NA

290

85Ye

s45

(9%

)0.

623

9/27

398

.8

14N

oYe

sN

o≥

39.4

°C6.

32.

7930

.866

64.

2428

012

4Ye

s34

(7%

)1.

122

4/33

797

.06

15N

oYe

sN

o<

38.4

°C8.

15

80.3

1759

3.28

110

57Ye

s43

(9%

)1.

618

5/33

774

.64

16N

oYe

sN

o≥

39.4

°C6

3.93

6069

005.

1210

012

9Ye

s40

(8%

)0.

530

4/33

799

.97

*g/d

L.†1

03 /µL.

‡ng/

mL.

§mm

ol/L

.¶m

g/dL

.**

U/L

.††

Nº h

istio

cyte

s pe

r 500

nuc

leat

ed c

ells.

‡‡Sc

ore

poin

ts/ m

axim

al p

oint

s fo

r thi

s pa

tient

with

his

/her

ava

ilabl

e pa

ram

eter

s.BM

, bon

e m

arro

w; G

OT, g

luta

mic

oxa

loac

etic

tran

sam

inas

e; H

b, h

aem

oglo

bin;

HPF

, hig

h po

wer

fiel

d; IS

, im

mun

osup

pres

sion

; NA,

not

ava

ilabl

e; W

BC, w

hite

cel

l cou

nt.

on Novem

ber 2, 2021 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jclinpath-2020-207337 on 15 M

arch 2021. Dow

nloaded from

5Núñez- Torrón C, et al. J Clin Pathol 2021;0:1–7. doi:10.1136/jclinpath-2020-207337

Original research

Lymph node tissue was collected from six patients. All of them displayed small size. Lymphoid depletion with sinusal histiocy-tosis was a common finding (5/6 patients). Haemophagocytosis was observed in four patients (figure 2). Three patients showed an extended interfollicular plasmocytosis.

HScoreParameters evaluated in each case and final HScore are described in table 2.

We could collect all HScore parameters in 10 patients (62.5%). In three patients, ferritin levels were missing (273/337 points were available), and in other three patients, triglycerides and ferritin levels were missing (223/337 points were available). Among the patients in which all parameters were evaluable, 8 patients (80%) had an HScore >169. Between the patients with a HScore <169, four were patients with missing data. sHLH was not clinically suspected in any case.

During COVID-19 evolution, a maximum body temperature >38·4°C was present in 12 patients (75%), splenomegaly in 1 patient (6·3%) and hepatomegaly in 4 patients (25%). Among laboratory parameters, 13 patients (81·3%) presented a haemo-globin level <9·2 g/dL, 10 patients (62·5%) a leucocyte count <5000×109/L, 13 patients (81·3%) platelets <110×109/L, 14 patients (87·5%) fibrinogen <250 mg/dL and all patients a maximum glutamic oxaloacetic transaminase (GOT) >30 U/L. Among the 10 patients with available triglyceride levels, hyper-triglyceridemia >1·5 mmol/L was present in 8 patients (80%) and among the 13 patients with ferritin available, ferritin level >2000 ng/mL was present in 4 patients (30·8%).

All patients presented haemophagocytosis in bone marrow tissue. From the eight patients with all HScore parameters avail-able and a total points 169, all but one displayed pathological (moderate to severe) HHH, six in moderate degree and one in severe degree. In all of them, more than one nucleated cell within a single haemophagocyte was observed. Of those eight patients, four of them would not reach a score of 169 without the item of haemophagocytosis in bone marrow.

DISCUSSIONThe differential diagnosis between a severe infection and HLH can be challenging since an infection can precede and trigger HLH.30 Furthermore, if there is not a high index of suspicion, the diagnosis of sHLH is often missed or delayed. Unless it is diagnosed early and an appropriate treatment is instituted, the mortality rate is high.31

It has been suggested that a subgroup of patients suffering from severe COVID-19 pneumonia could develop sHLH.2 32 33 These patients meet the laboratory parameters necessary to the diagnosis of sHLH such as elevated CPR and hyperferritinemia and other clinical conditions including abnormal liver function and coagulopathy.34 Previous related viruses as SARS- CoV and MERS- CoV showed similar clinical course than SARS- CoV-2, although in the first two, the proportion of patients with fatal evolution was higher, especially in MERS- COV.35

We calculated the HScore of 16 patients with COVID-19. In 10 of these patients (62·5%), all the parameters could be collected and HScore was evaluable. Among these, 80% had a global score >169 points. In those patients in which HScore was lower, in 66% not all parameters could be collected. Our results support the recommendation of some authors of suspecting hyperinflammation in patients with severe COVID-19 and using HScore in order to identify patients who could benefit from immunosuppressive therapies.18 Haemophagocytic syndrome was not clinically suspected in any of our cases.

Levernez et al preclude that HScore is not reliable in patients with COVID-19 due to its lack of specificity when assessing the leukocytopenia as it does not differentiate neutropenia from lymphopenia and the absence of extremely elevated ferritin levels in patients with COVID-19.36 It is true that patients with COVID-19 have some differences with patients with sHLH, being the most important the absence of lymphadenopathy or splenomegaly, the absence of hypofibrinogenemia and the not so extremely elevated ferritin levels.3 4 19 However, based on our findings and previous reports, we consider that they share core similarities (presence of haemophagocytosis, raised ferritin levels) which reflect a common immune response in COVID-19 and sHLH driven by the macrophages. Moreover, the only treatment for COVID-19 with impact on mortality to date (dexamethasone) is one of the main agents included in HLH protocols.37 38 We consider that HScore is a useful tool to iden-tify this sHLH- like syndrome in COVID-19 patients.

McGonagle et al,34 based on an immunopathology model, postulated that sHLH in COVID-19 pneumonia could be restricted to the pulmonary compartment, being considered an acute ARDS damage rather than a systemic macrophage activa-tion, characteristic of sHLH. In our study, we found that not only pulmonary compartment was involved, but also reticuloendo-thelial organs. Therefore, our results contradict this hypothesis.

Regarding the presence of haemophagocytosis in bone marrow tissue, it has been reported that most of the patients have haemophagocytosis at the time of diagnosis of HLH.39 Previous studies have attempted to establish the number of haemophagocytosis in bone marrow that could define a diag-nosis of HLH.20 31 40–43 Although there is not a consensus, the probability may be higher if severe haemophagocytosis is observed.20 Gars et al29 evaluated the presence of multiple nucle-ated cells within a single haemophagocyte in bone marrow as a possible morphological feature that may differentiate patients with pathological haemophagocytosis. In our study, the presence of multiple nuclei within the same histiocyte seems to be more related to the grade of HHH rather than to sHLH. It is known

Figure 2 Lymph node tissue pathological findings. (A) (H&E stain, 4x) Low- power of view lymph node in which sinusal histiocytosis is remarkable. (B) (H&E stain, 10x) Low- power view of sinusal histiocytosis in lymph node. (C) (CD68 stain, 4x) Low- power of view of lymph node with sinusal histiocytosis. (D) (CD68 stain, 40x) High- power view of haemophagocytosis in lymph node.

on Novem

ber 2, 2021 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jclinpath-2020-207337 on 15 M

arch 2021. Dow

nloaded from

6 Núñez- Torrón C, et al. J Clin Pathol 2021;0:1–7. doi:10.1136/jclinpath-2020-207337

Original research

that the demonstration on bone marrow aspiration/biopsy is not mandatory for the diagnosis of sHLH.44 45 Nevertheless, it can be a useful tool, if the other characteristic criteria are not avail-able in time to aid in immediate treatment- related decisions.31 In our study, this parameter has been decisive at the time of calcu-lating HScore in many patients46 besides this phenomenon is not specific of HLH, as it can be seen in other conditions such as postblood transfusion, haemolysis, myelodysplasia/bone marrow failure or sepsis.44–49 Therefore, it is mandatory to exclude all these conditions when haemophagocytosis is observed and HLH is suspected.

Few studies of COVID-19 autopsy series21 23 24 have demonstrated the presence of haemophagocytosis in bone marrow in some of the patients. Most of them are based on small series or samples obtained from bone marrow aspiration or trephine biopsy. We present one of the largest autopsy series focusing on the study of very representative samples of bone marrow, taken from rib squeeze. Moreover, quanti-fication of the grade of HHH and its correlation with the clinical and analytical features distinctive of HLHs HScore have not been done until the present study. Although haemophagocytosis was present in all the studied cases, meeting the pathological parameter of the HScore, our results show that the patients with higher probability of sHLH displayed severe or moderate HHH.

Regarding the limitations of the study, it must be considered that the clinical and analytical parameters taken from our patients corre-spond to their highest values, not to the time when they worsen clini-cally, since they were studied postmortem. Based on this observation, we recommend, when sHLH is suspected, to collect the laboratory and clinical parameters on a standardised basis.

In conclusion, in virtue of the high percentage of cases (80% of the evaluable cases) that had a global HScore >169 points observed in our study, the possibility of HLH secondary to COVID-19 must be considered, especially in severe patients. Therefore, it would be advisable to calculate the HScore in these patients in order to identify patients who may benefit from more intensified immunosuppressive therapy.

Bone marrow haemophagocytosis is frequent and, therefore, HLH may be underestimated when H- scores are calculated without a bone marrow biopsy, so in those patients with clinical suspicion of sHLH in which HScore is close to the 169 cut- off, bone marrow biopsy with immunohistochemical staining for histiocytic markers may aid in establishing the sHLH diagnosis.

Take home messages

► We are presenting one of the largest autopsy series demonstrating haemophagocytosis in bone marrow, added to the contribution of clinical data to integrate a possible diagnosis of secondary haemophagocytic lymphohistiocytosis (sHLH) in patients with COVID-19.

► All 16 patients had any degree of histiocytichyperplasia with haemophagocytosis in the bone marrow sample. Among evaluable patients for HScore, 80% had >169 points.

► In severe patients with COVID-19, it would be advisable to calculate the HScore in order to identify who may benefit from more intensified immunosuppressive therapy.

► In cases with total points close to 169, a bone marrow sample could help to establish the sHLH diagnosis.

Handling editor Mary Frances McMullin.

Contributors AF- G and CN- T have contributed equally as principal investigator. MG- C and MP- V have contributed equally as senior author. AF- G, EMM, BP- M, JP and MG- C participated in the autopsy procedure. CN- T, AF- G, MP- V and MG- C

recollected the data. CN- T, AF- G, MP- V, MG- C and JP drafted the manuscript. All authors contributed to the final approved version of this manuscript.

Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not- for- profit sectors.

Competing interests None declared.

Patient consent for publication Not required.

Ethics approval This work has been reviewed and approved by the Ramón y Cajal Hospital Ethics Committee.

Provenance and peer review Not commissioned; externally peer reviewed.

Data availability statement All data relevant to the study are included in the article.

This article is made freely available for use in accordance with BMJ’s website terms and conditions for the duration of the covid-19 pandemic or until otherwise determined by BMJ. You may use, download and print the article for any lawful, non- commercial purpose (including text and data mining) provided that all copyright notices and trade marks are retained.

ORCID iDClaudia Núñez- Torrón http:// orcid. org/ 0000- 0002- 2881- 161X

REFERENCES 1 Mizumoto K, Kagaya K, Zarebski A, et al. Estimating the asymptomatic proportion of

coronavirus disease 2019 (COVID-19) cases on board the diamond Princess cruise SHIP, Yokohama, Japan, 2020. Eurosurveillance 2020;25:2000180.

2 Quan C, Li C, Ma H, et al. Immunopathogenesis of coronavirus- induced acute respiratory distress syndrome (ARDS): potential infection- associated hemophagocytic lymphohistiocytosis. Clin Microbiol Rev 2020;34:e00074–20.

3 Huang C, Wang Y, Li X, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. The Lancet 2020;395:497–506.

4 Zhou F, Yu T, Du R, et al. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. The Lancet 2020;395:1054–62.

5 Yang X, Yu Y, Xu J, et al. Clinical course and outcomes of critically ill patients with SARS- CoV-2 pneumonia in Wuhan, China: a single- centered, retrospective, observational study. Lancet Respir Med 2020;8:475–81.

6 Liu B, Li M, Zhou Z, et al. Can we use interleukin-6 (IL-6) blockade for coronavirus disease 2019 (COVID-19)- induced cytokine release syndrome (CRS)? J Autoimmun 2020;111:102452.

7 Giamarellos- Bourboulis EJ, Netea MG, Rovina N, et al. Complex immune dysregulation in COVID-19 patients with severe respiratory failure. Cell Host Microbe 2020;27:992–1000.

8 Dimopoulos G, de Mast Q, Markou N, et al. Favorable Anakinra responses in severe Covid-19 patients with secondary hemophagocytic lymphohistiocytosis. Cell Host Microbe 2020;28:117–23.

9 Kumar B, Aleem S, Saleh H, et al. A personalized diagnostic and treatment approach for macrophage activation syndrome and secondary hemophagocytic lymphohistiocytosis in adults. J Clin Immunol 2017;37:638–43.

10 Usmani GN, Woda BA, Newburger PE. Advances in understanding the pathogenesis of hLH. Br J Haematol 2013;161:609–22.

11 Lee DW, Gardner R, Porter DL, et al. Current concepts in the diagnosis and management of cytokine release syndrome. Blood 2014;124:188–95.

12 Schram AM, Berliner N. How I treat hemophagocytic lymphohistiocytosis in the adult patient. Blood 2015;125:2908–14.

13 Henter J- I, Chow C- B, Leung C- W, et al. Cytotoxic therapy for severe avian influenza A (H5N1) infection. Lancet 2006;367:870–3.

14 Esteban YM, de Jong JLO, Tesher MS. An overview of hemophagocytic lymphohistiocytosis. Pediatr Ann 2017;46:e309–13.

15 Lerolle N, Laanani M, Rivière S, et al. Diversity and combinations of infectious agents in 38 adults with an infection- triggered reactive haemophagocytic syndrome: a multicenter study. Clin Microbiol Infect 2016;22:268.e1–268.e8.

16 Henter J- I, Horne A, Aricó M, et al. HLH-2004: diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer 2007;48:124–31.

17 Fardet L, Galicier L, Lambotte O, et al. Development and validation of the HScore, a score for the diagnosis of reactive hemophagocytic syndrome. Arthritis & Rheumatology 2014;66:2613–20.

18 Mehta P, McAuley DF, Brown M, et al. COVID-19: consider cytokine storm syndromes and immunosuppression. Lancet 2020;395:1033–4.

19 Wood H, Jones JR, Hui K, et al. Secondary HLH is uncommon in severe COVID-19. Br J Haematol 2020;190:e283–5.

20 Gupta A, Weitzman S, Abdelhaleem M. The role of hemophagocytosis in bone marrow aspirates in the diagnosis of hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer 2008;50:192–4.

21 Prilutskiy A, Kritselis M, Shevtsov A, et al. SARS- CoV-2 infection- associated hemophagocytic lymphohistiocytosis. Am J Clin Pathol 2020;154:466–74.

on Novem

ber 2, 2021 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jclinpath-2020-207337 on 15 M

arch 2021. Dow

nloaded from

7Núñez- Torrón C, et al. J Clin Pathol 2021;0:1–7. doi:10.1136/jclinpath-2020-207337

Original research

22 Dewaele K, Claeys R. Hemophagocytic lymphohistiocytosis in SARS- CoV-2 infection. Blood 2020;135:2323.

23 Prieto- Pérez L, Fortes J, Soto C, et al. Histiocytic hyperplasia with hemophagocytosis and acute alveolar damage in COVID-19 infection. Mod Pathol 2020;33:2139–46.

24 Bryce C, Grimes Z, Pujadas E. Pathophysiology of SARS- CoV-2: targeting of endothelial cells renders a complex disease with thrombotic microangiopathy and aberrant immune response. The Mount Sinai COVID-19 autopsy experience. In press.

25 Hanley B, Naresh KN, Roufosse C, et al. Histopathological findings and viral tropism in UK patients with severe fatal COVID-19: a post- mortem study. Lancet Microbe 2020;1:e245–53.

26 Jin Y- H, Cai L, Cheng Z- S, et al. A rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019- nCoV) infected pneumonia (standard version). Mil Med Res 2020;7:4.

27 COVID-19 Autopsy. Electronic address: anapat. hrc@ salud. madrid. org. The first COVID-19 autopsy in Spain performed during the early stages of the pandemic. Rev Esp Patol 2020;53:182–7.

28 Suster S, Hilsenbeck S, Rywlin AM. Reactive histiocytic hyperplasia with hemophagocytosis in hematopoietic organs: a reevaluation of the benign hemophagocytic proliferations. Hum Pathol 1988;19:705–12.

29 Gars E, Purington N, Scott G, et al. Bone marrow histomorphological criteria can accurately diagnose hemophagocytic lymphohistiocytosis. Haematologica 2018;103:1635–41.

30 Karlsson T. Secondary haemophagocytic lymphohistiocytosis: experience from the Uppsala university hospital. Ups J Med Sci 2015;120:257–62.

31 Nair V, Das S, Sharma A, et al. A clinicopathological analysis of 26 patients with infection- associated haemophagocytic lymphohistiocytosis and the importance of bone marrow phagocytosis for the early initiation of immunomodulatory treatment. Postgrad Med J 2013;89:185–92.

32 Bracaglia C, Prencipe G, De Benedetti F. Macrophage activation syndrome: different mechanisms leading to a one clinical syndrome. Pediatr Rheumatol Online J 2017;15:5.

33 Wu C, Chen X, Cai Y, et al. Risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in Wuhan, China. JAMA Intern Med 2020;180:934–43.

34 McGonagle D, Sharif K, O’Regan A, et al. The role of cytokines including interleukin-6 in COVID-19 induced pneumonia and macrophage activation syndrome- like disease. Autoimmun Rev 2020;19:102537.

35 Tisoncik JR, Korth MJ, Simmons CP, et al. Into the eye of the cytokine storm. Microbiol Mol Biol Rev 2012;76:16–32.

36 Leverenz DL, Tarrant TK. Is the HScore useful in COVID-19? Lancet 2020;395:e83. 37 Horby P, Lim WS, et al, RECOVERY Collaborative Group. Dexamethasone in

Hospitalized Patients with Covid-19 - Preliminary Report. N Engl J Med 2020. [Epub ahead of print: 17 Jul 2020]. doi:10.1056/NEJMoa2021436

38 Bergsten E, Horne A, Aricó M, et al. Confirmed efficacy of etoposide and dexamethasone in HLH treatment: long- term results of the cooperative HLH-2004 study. Blood 2017;130:2728–38.

39 Weitzman S, Jaffe R. Uncommon histiocytic disorders: the non- Langerhans cell histiocytoses. Pediatr Blood Cancer 2005;45:256–64.

40 Risdall RJ, McKenna RW, Nesbit ME, et al. Virus- Associated hemophagocytic syndrome: a benign histiocytic proliferation distinct from malignant histiocytosis. Cancer 1979;44:993–1002.

41 Favara BE. Hemophagocytic lymphohistiocytosis: a hemophagocytic syndrome. Semin Diagn Pathol 1992;9:63–74.

42 Marmont AM, Spriano M. Hemophagocytic lymphohistiocytosis: still a morphological diagnosis. Haematologica. October 1995;80:480–1.

43 zur Stadt U, Schmidt S, Kasper B, et al. Linkage of familial hemophagocytic lymphohistiocytosis (FHL) type-4 to chromosome 6q24 and identification of mutations in syntaxin 11. Hum Mol Genet 2005;14:827–34.

44 Lehmberg K, Ehl S. Diagnostic evaluation of patients with suspected haemophagocytic lymphohistiocytosis. Br J Haematol 2013;160:275–87.

45 Raschke RA, Garcia- Orr R. Hemophagocytic lymphohistiocytosis: a potentially underrecognized association with systemic inflammatory response syndrome, severe sepsis, and septic shock in adults. Chest 2011;140:933–8.

46 Henter J- I, Samuelsson- Horne A, Aricò M, et al. Treatment of hemophagocytic lymphohistiocytosis with HLH-94 immunochemotherapy and bone marrow transplantation. Blood 2002;100:2367–73.

47 Jordan MB, Allen CE, Weitzman S, et al. How I treat hemophagocytic lymphohistiocytosis. Blood 2011;118:4041–52.

48 Canna SW, Behrens EM. Not all hemophagocytes are created equally: appreciating the heterogeneity of the hemophagocytic syndromes. Curr Opin Rheumatol 2012;24:113–8.

49 Okabe T, Shah G, Mendoza V, et al. What intensivists need to know about hemophagocytic syndrome: an underrecognized cause of death in adult intensive care units. J Intensive Care Med 2012;27:58–64.

on Novem

ber 2, 2021 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jclinpath-2020-207337 on 15 M

arch 2021. Dow

nloaded from